Determination of hammerhead ribozyme kinetic constants at high molar ratio ribozyme-substrate.

Hammerhead ribozymes provide valuable tools in the field of gene therapy due to their cleavage specificity and the broad range of RNA targets. A major prerequisite for the selection of suitable ribozymes for in vivo application is represented by in vitro determination of ribozyme cleavage kinetic constants. From the experimental cleavage data, kinetic constants are usually calculated under the assumption of rapid conversion of the substrate into the ribozyme-substrate complex. However, this condition is often not satisfied for ribozymes carrying additional RNA stretches, due to cloning strategies or necessary for ribozyme expression in the cell. To overcome this problem, we propose a mathematical model which is able to calculate ribozyme kinetic constants in the case of non-rapid conversion of substrate into ribozyme-substrate complex. In addition, our system gives the opportunity to evaluate the nature of the S conversion into ES through the determination of a model parameter. The validity of the proposed model is restricted to the hypothesis of a ribozyme excess over the substrate at the beginning of the cleavage reaction and to the absence of any mass exchange with the external environment.